A bispecific antibody (BsAb), or specific monoclonal antibody (BsMAb) is an artificial protein comprised of fragments from two different antibodies. By virtue of the inclusion of two different antigen binding regions, a bispecific antibody is capable of recognizing and binding to two different antigens, or two different epitope on an antigen. In cancer immunotherapy, bispecific antibodies are being developed to simultaneously bind to a cytotoxic cell by targeting a receptor like CD3, and a tumor cell to be destroyed.
Bispecific antibodies present challenges in various aspects. First, they are more difficult to manufacture. Further, the in vitro and in vivo stabilities of these artificial proteins may be questionable. Given the close proximity of the antigen binding regions within a single protein, it also remains to be seen whether the antigen binding regions retain their binding affinities.
In order to overcome manufacturing difficulties, a first-generation BsMAb, called trifunctional antibody, has been developed. It consists of two heavy and two light chains, one each from two different antibodies. The two Fab regions are directed against two antigens. The Fc region (the foot) is made up from the two heavy chains and forms the third binding site; hence the name.
Other types of bispecific antibodies have been designed to overcome certain problems, such as short half-life, immunogenicity and side-effects caused by cytokine liberation. They include chemically linked Fabs, consisting only of the Fab regions, and various types of bivalent and trivalent single-chain variable fragments (scFvs), fusion proteins mimicking the variable domains of two antibodies.
Other types of recombinant antibody formats have also been developed in the recent past, e.g. tetravalent bispecific antibodies by fusion of, e.g., an IgG antibody format and single chain domains (see e.g. Coloma, M. J., et al., Nature Biotech 15 (1997) 159-163; WO 2001/077342; and Morrison, S. L., Nature Biotech 25 (2007) 1233-1234).
Also several other new formats wherein the antibody core structure (IgA, IgD, IgE, IgG or IgM) is no longer retained such as dia-, tria- or tetrabodies, minibodies, several single chain formats (scFv, Bis-scFv), which are capable of binding two or more antigens, have been developed (Holliger, P., et al., Nature Biotech 23 (2005) 1126-1136; Fischer, N., Leger, O., Pathobiology 74 (2007) 3-14; Shen, J., et al., Journal of Immunological Methods 318 (2007) 65-74; Wu, C., et al., Nature Biotech. 25 (2007) 1290-1297).
All such formats use linkers either to fuse the antibody core (IgA, IgD, IgE, IgG or IgM) to a further binding protein (e.g. scFv) or to fuse e.g. two Fab fragments or scFvs (Fischer, N., Leger, O., Pathobiology 74 (2007) 3-14). It is important to retain effector functions, such as e.g. complement-dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC), which are mediated through the Fc receptor binding, by maintaining a high degree of similarity to naturally occurring antibodies.
WO 2007/024715 reports dual variable domain immunoglobulins as engineered multivalent and multispecific binding proteins. A process for the preparation of biologically active antibody dimers is reported in U.S. Pat. No. 6,897,044. Multivalent Fv antibody construct having at least four variable domains which are linked with each other via peptide linkers are reported in U.S. Pat. No. 7,129,330. Dimeric and multimeric antigen binding structures are reported in US 2005/0079170. Tri- or tetra-valent monospecific antigen-binding protein comprising three or four Fab fragments bound to each other covalently by a connecting structure, which protein is not a natural immunoglobulin are reported in U.S. Pat. No. 6,511,663. In WO 2006/020258 tetravalent bispecific antibodies are reported that can be efficiently expressed in prokaryotic and eukaryotic cells, and are useful in therapeutic and diagnostic methods. A method of separating or preferentially synthesizing dimers which are linked via at least one interchain disulfide linkage or dimers which are not linked via at least one interchain disulfide linkage from a mixture comprising the two types of polypeptide dimers is reported in US 2005/0163782. Bispecific tetravalent receptors are reported in U.S. Pat. No. 5,959,083. Engineered antibodies with three or more functional antigen binding sites are reported in WO 2001/077342.
Multispecific and multivalent antigen-binding polypeptides are reported in WO 1997/001580. WO 1992/004053 reports homoconjugates, typically prepared from monoclonal antibodies of the IgG class which bind to the same antigenic determinant are covalently linked by synthetic cross-linking Oligomeric monoclonal antibodies with high avidity for antigen are reported in WO 1991/06305 whereby the oligomers, typically of the IgG class, are secreted having two or more immunoglobulin monomers associated together to form tetravalent or hexavalent IgG molecules. Sheep-derived antibodies and engineered antibody constructs are reported in U.S. Pat. No. 6,350,860, which can be used to treat diseases wherein interferon gamma activity is pathogenic. In US 2005/0100543 are reported targetable constructs that are multivalent carriers of bi-specific antibodies, i.e., each molecule of a targetable construct can serve as a carrier of two or more bi-specific antibodies. Genetically engineered bispecific tetravalent antibodies are reported in WO 1995/009917. In WO 2007/109254 stabilized binding molecules that consist of or comprise a stabilized scFv are reported.
Epidermal growth factor receptor (EGFR) is a trans-membrane receptor encoded by the c-erbB 1 proto-oncogene with a molecular weight of approximately 170 kDa. EGFR is normally expressed in a wide variety of epithelial tissues as well as in the central nervous system. Accumulating evidence suggests that the level of EGFR overexpression is an important factor that directly correlates with active proliferation of malignant cells and poor prognosis of patients, thus, providing the rationale for the development of EGFR antagonists as potentially useful therapeutic strategies for the treatment of EGFR-expressing cancers.
EGFR inhibitors encompassing both small molecules and antibodies have been developed for the treatment of cancer. The small-molecule EGFR tyrosine kinase inhibitors (TKI) erlotinib (Tarceva®) and gefitinib (Iressa®) have demonstrated activity in multiple epithelial tumor types. These compounds reversibly bind to the adenosine triphosphate binding site of the EGFR TKD and inhibit autophosphorylation. Initial results with these molecules as monotherapy or in combination with chemotherapy in unselected populations were disappointing. It is now known that mutations in the EGFR gene alter the tumur phenotype and predict response to treatment, allowing the molecular selection of a subset of patients in which TKI are highly efficacious. The anti-EGFR monoclonal antibodies (mAbs) cetuximab (Erbitux®) and panitumumab (Vectibix®) are established agents in the treatment of CRC (colon and rectal cancer) and SCCHN (Squamous Cell Carcinoma of the Head and Neck). These agents have demonstrated modest clinical efficacy in combination with chemotherapy in phase III trials. However, patients with CRC with KRAS mutations (30%-40% of patients) are unresponsive to cetuximab or panitumumab, when used as monotherapy or in combination with chemotherapy. mAbs targeting cell surface receptors can exert a therapeutic effect either by inhibiting the oncogenic growth signal (blocking ligand binding and/or receptor dimerisation/activation) or through direct cell killing. Cell killing can be achieved by inducing apoptosis in the target cell or cell killing can be achieved by releasing cytotoxic compounds in the target cell through antibody-drug conjugates (ADCs), which consist of cytotoxic agents or toxins chemically conjugated to a monoclonal antibody. Antibody-drug conjugates potentially represent an advantage over treatment with chemotherapy because they are designed to deliver the cytotoxic agent specifically to tumor cells thereby resulting in an improved safety profile.
Angiogenesis is involved in the pathogenesis of a variety of diseases, including solid tumors, intraocular neovascular syndromes such as proliferative retinopathies or age-related macular degeneration (AMD), rheumatoid arthritis, and psoriasis (Folkman, J., et al., J. Biol. Chem. 267 (1992) 10931-10934; Klagsbrun, M., et al., Annu Rev. Physiol. 53 (1991) 217-239). Angiogenesis allows the tumor cells to acquire a growth advantage and proliferative autonomy compared to the normal cells. Human vascular endothelial growth factor (VEGF/VEGF-A) is involved in the regulation of normal and abnormal angiogenesis and neovascularization associated with tumors and intraocular disorders (Ferrara, N., et al., Endocr. Rev. 18 (1997) 4-25; Berkman, R. A., et al., J. Clin. Invest. 91 (1993) 153-159; Brown, L. F., et al., Human Pathol. 26 (1995) 86-91; Brown, L. F., et al., Cancer Res. 53 (1993) 4727-4735; Mattern, J., et al., Brit. J. Cancer. 73 (1996) 931-934; and Dvorak, H., et al., Am. J. Pathol. 146 (1995) 1029-1039).
VEGF is a homodimeric glycoprotein that has been isolated from several sources. VEGF shows highly specific mitogenic activity for endothelial cells. VEGF has important regulatory functions in the formation of new blood vessels during embryonic vasculogenesis and in angiogenesis during adult life (Carmeliet, P., et al., Nature, 380 (1996) 435-439; Ferrara, N., et al., Nature, 380 (1996) 439-442; reviewed in Ferrara and Davis-Smyth, Endocrine Rev., 18 (1997) 4-25. The significance of the role played by VEGF has been demonstrated in studies showing that inactivation of a single VEGF allele results in embryonic lethality due to failed development of the vasculature (Carmeliet, P., et al., Nature, 380 (1996) 435-439; Ferrara, N., et al., Nature, 380 (1996) 439-442.
In addition VEGF has strong chemoattractant activity towards monocytes, can induce the plasminogen activator and the plasminogen activator inhibitor in endothelial cells, and can also induce microvascular permeability. Because of the latter activity, it is sometimes referred to as vascular permeability factor (VPF). The isolation and properties of VEGF have been reviewed; see Ferrara, N., et al., J. Cellular Biochem., 47 (1991) 211-218 and Connolly, J. Cellular Biochem., 47 (1991) 219-223. Alternative mRNA splicing of a single VEGF gene gives rise to five isoforms of VEGF.
Anti-VEGF neutralizing antibodies suppress the growth of a variety of human tumor cell lines in mice (Kim, I., et al., Nature 362 (1993) 841-844; Warren, S. R., et al., J. Clin. Invest. 95 (1995) 1789-1797; Borgstrom, P., et al., Cancer Res. 56 (1996) 4032-4039; and Melnyk, O., et al., Cancer Res. 56 (1996) 921-924). WO 94/10202, WO 98/45332, WO 2005/00900 and WO 00/35956 refer to antibodies against VEGF. Humanized monoclonal antibody bevacizumab (sold under the trade name Avastin®) is an anti-VEGF antibody used in tumor therapy (WO 98/45331).
Bevacizumab, combined with fluoropyrimidine-based chemotherapy is now the standard first-line treatment for metastatic colorectal cancer. Cetuximab, a chimeric IgG1 monoclonal antibody against epidermal growth factor receptor (EGFR), has efficacy as monotherapy and in combination with irinotecan in irinotecan-resistant patients. It would have been expected that the addition of cetuximab to capecitabine, oxaliplatin, and bevacizumab as first-line treatment in patients with metastatic colorectal cancer (the CAIRO2 trial) would achieve better efficacy by blocking both EGFR and VEGF.
However, overall survival and response rates did not improve in the treatment group of combing the two blocking antibodies, cetuximab and bevacizumab. Patients treated with cetuximab who had tumors bearing a mutated KRAS gene had significantly decreased progression-free survival (PFS) as compared with cetuximab-treated patients with wildtype-KRAS tumors (N Engl J Med 2009; 360:563-72).
Panitumumab, a fully human antibody targeting the epidermal growth factor receptor, approved for treatment for patients with metastatic colorectal cancer (mCRC), was evaluated in another trial by adding to bevacizumab and chemotherapy (oxaliplatin- and irinotecan-based) as first-line treatment for mCRC, similar data were obtained. The addition of panitumumab to bevacizumab and oxaliplatin- or irinotecan-based chemotherapy results in increased toxicity and decreased PFS. These simple drug combinations are not recommended for the treatment of mCRC in clinical practice (J Clin Oncol (2009) 27:672-680). Thus, there is an apparent need to a better approach to the treatment of solid tumors by targeting therapeutic agents to the tumor tissues, while simultaneously blocking the signaling pathway by both VEGF and EGFR.
Human domain antibodies selected against VEGF and EGFR were reported by formatting into a dual-targeting IgG (DT-IgG) to directly target both antigens in a single molecule (Int J Cancer. 2012 Aug. 15; 131(4):956-69). This DT-IgG suppressed EGFR positive cell growth, inhibited EGFR activation and induced apoptosis as effectively as cetuximab, and neutralized VEGF as effectively as bevacizumab. However, DT-IgG format is single domain based, and have a very short half-life, and thus is not suitable as therapeutic treatment for cancers.